Plant lipids have a variety of industrial and nutritional uses and are central to plant membrane function and climatic adaptation. These lipids represent a vast array of chemical structures, and these structures determine the physiological and industrial properties of the lipid. Many of these structures result either directly or indirectly from metabolic processes that alter the degree of unsaturation of the lipid. Different metabolic regimes in different plants produce these altered lipids, and either domestication of exotic plant species or modification of agronomically adapted species is usually required to economically produce large amounts of the desired lipid.
Plant lipids find their major use as edible oils in the form of triacylglycerols. The specific performance and health attributes of edible oils are determined largely by their fatty acid composition. Most vegetable oils derived from commercial plant varieties are composed primarily of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acids. Palmitic and stearic acids are, respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic, linoleic, and linolenic acids are 18-carbon-long, unsaturated fatty acids containing one, two, and three double bonds, respectively. Oleic acid is referred to as a mono-unsaturated fatty acid, while linoleic and linolenic acids are referred to as poly-unsaturated fatty acids. The relative amounts of saturated and unsaturated fatty acids in commonly used, edible vegetable oils are summarized below (Table 1).
TABLE 1Percentages of Saturated and Unsaturated FattyAcids in the Oils of Selected Oil CropsSaturatedMono-unsaturatedPoly-unsaturatedCanola 6%58%36%Soybean15%24%61%Corn13%25%62%Peanut18%48%34%Safflower 9%13%78%Sunflower 9%41%51%Cotton30%19%51%
Many recent research efforts have examined the role that saturated and unsaturated fatty acids play in reducing the risk of coronary heart disease. In the past, it was believed that mono-unsaturates, in contrast to saturates and poly-unsaturates, had no effect on serum cholesterol and coronary heart disease risk. Several recent human clinical studies suggest that diets high in mono-unsaturated fat and low in saturated fat may reduce the “bad” (low-density lipoprotein) cholesterol while maintaining the “good” (high-density lipoprotein) cholesterol (Mattson et al. (1985) Journal of Lipid Research 26:194-202).
A vegetable oil low in total saturates and high in mono-unsaturates would provide significant health benefits to consumers as well as economic benefits to oil processors. As an example, canola oil is considered a very healthy oil. However, in use, the high level of poly-unsaturated fatty acids in canola oil renders the oil unstable, easily oxidized, and susceptible to development of disagreeable odors and flavors (Gailliard (1980) in The Biochemistry of Plants Vol. 4, pp. 85-116, Stumpf, P. K., ed., Academic Press, New York). The levels of poly-unsaturates may be reduced by hydrogenation, but the expense of this process and the concomitant production of nutritionally questionable trans isomers of the remaining unsaturated fatty acids reduces the overall desirability of the hydrogenated oil (Mensink et al. (1990) New England J. Medicine N323: 439-445). Similar problems exist with soybean oil and as noted in Table 1, commodity soybean oil typically contains over twice the saturated fat content of canola oil.
Mutation-breeding programs have met with some success in altering the levels of poly-unsaturated fatty acid levels found in the edible oils of agronomic species. Examples of commercially grown varieties are high (85%) oleic sunflower and low (2%) linolenic flax (Knowles (1980) in World Conference on Biotechnology for the Fats and Oils Industry Proceedings, Applewhite, T. H., ed., American Oil Chemists' Society, pp. 35-38). Similar commercial progress with the other plants shown in Table 1 has been elusive, largely due to the difficult nature of the procedure and the pleiotropic effects of the mutational regime on plant hardiness, yield potential and the environmental instability of the low poly-unsaturate trait. Above all, the inability to consistently produce an oil of defined composition from season to season and in differing locations has made the commercial production of low poly-unsaturate soybean oil infeasible.
The discovery of a method for altering the expression of the enzymes responsible for introduction of the second (international patent publication WO 94/11516) and third (international patent publication WO 93/11245) double bonds into soybean seed storage lipid in a directed manner has allowed the production of soybeans with a high mono-unsaturated, very low polyunsaturated fatty acid content and especially a very low linolenic acid content. The genetic combination of these two transgene profiles described in the instant invention leads to a soybean line with minimal poly-unsaturates and high mono-unsaturates and extreme environmental stability of the seed fatty acid profile.
Soybeans with decreased levels of saturated fatty acids have been described resulting from mutation breeding (Erickson, E. A. et al., (1994) J. Hered. 79:465-468; Schnebly, S. R. et al. (1994) Crop Sci. 34:829-833; and Fehr, W. R. et al. (1991) Crop Sci. 31:88-89) and transgenic modification (U.S. Pat. No. 5,530,186). The demonstration of the combination of these two traits in a single soybean line in this invention brings the health benefits of a high mono-unsaturate, low saturate oil to soybean production.
While oils with low levels of saturated fatty acids are desirable from the standpoint of providing a healthy diet, fats that are solid at room temperature are required for their functional properties in some foods. Such applications include the production of non-dairy margarines and spreads, and various applications in confections and in baking. Many animal and dairy fats provide the necessary physical properties, but they also contain both cholesterol and the cholesterogenic medium chain fatty acids. An ideal triglyceride for solid fat applications should contain a predominance of the very high melting, long chain fatty acid stearic acid and a balance of mono-unsaturated fatty acid with very little polyunsaturated fat. Natural plant solid fat fractions typically have a triacylglyceride structure with saturated fatty acids occupying the sn-1 and sn-3 positions of the triglycerides and an unsaturated fatty acid at the sn-2 position. This overall fatty acid composition and triglyceride structure confers an optimal solid fat crystal structure and a maximum melting point with minimal saturated fatty acid content.
The natural fat prototype for this high melting temperature vegetable fat is cocoa butter. The fatty acid composition of cocoa butter is 26% palmitic (16:0), 34% stearic (18:0), 35% oleic (18:1), and 3% linoleic(18:2) acids. This fatty acid profile gives cocoa butter a melting point range of from 25° to 36° C. depending upon its precise crystal structure. The high price and fluctuating supply of cocoa butter has led to several processes for the production of cocoa butter substitutes and margarine stocks by fractionating other oils with relatively high 18:0 content or catalytic hydrogenation of high polyunsaturated oils followed by fractionation of the product.
Oilseeds capable of producing directly high stearate, low polyunsaturate oils would be advantageous in that both the cost of hydrogenation and the undesirable side products of hydrogenation, trans monounsaturated fatty acids, could be avoided. In addition, the fractionation process could be made more cost effective or possibly eliminated if the melting temperature range of the vegetable fat produced were high enough.
Oil biosynthesis in plants has been fairly well-studied [see Harwood (1989) in Critical Reviews in Plant Sciences, Vol. 8 (1):1-43]. The biosyntheses of palmitic, stearic and oleic acids occur in the plastids by the interplay of three key enzymes of the “ACP track”: palmitoyl-ACP elongase, stearoyl-ACP desaturase and the acyl-ACP thioesterases.
Of these three enzyme types, the acyl-ACP thioesterases function to remove the acyl chain from the carrier protein (ACP) and thus away from the metabolic pathway. Oleoyl-ACP thioesterase catalyzes the hydrolysis of oleoyl-ACP thioesters at relatively high rates, although it also catalyzes the hydrolysis of palmitoyl-ACP and stearoyl-ACP at much lower rates. This multiple activity leads to substrate competition between enzymes, and it is the competitions of acyl-ACP thioesterase and palmitoyl-ACP elongase for the same substrate and of acyl-ACP thioesterase and stearoyl-ACP desaturase for the same substrate that contributes to the production of the palmitic and stearic acids found in the triacylglycerides of vegetable oils.
Once removed from the ACP track, fatty acids are exported to the cytoplasm and there they are used to synthesize acyl-coenzyme A. These acyl-CoAs are the acyl donors for at least three different glycerol acylating enzymes (glycerol-3-P acyltransferase, 1-acyl-glycerol-3-P acyltransferase and diacylglycerol acyltransferase) which incorporate the acyl moieties into triacylglycerides during oil biosynthesis.
These acyltransferases show a strong, but not absolute, preference for incorporating saturated fatty acids at sn-1 and sn-3 positions and monounsaturated fatty acids at the sn-2 position of the triglyceride. Thus, altering the fatty acid composition of the acyl pool will drive a corresponding change in the fatty acid composition of the oil. Furthermore, there is experimental evidence that because of this specificity, plants can produce cocoa butter substitutes or other specialty fats if there is the correct composition of fatty acids available in the substrate pool for the acyltransferases [Bafor et al., (1990) JAOCS 67:217-225].
Based on the above discussion, one approach to changing the levels of palmitic, stearic and oleic acids in vegetable oils is to alter their levels in the cytoplasmic acyl-CoA pool used for oil biosynthesis.
Manipulation of stearate levels has been described (Knutzon, D. S. et al., (1992) Proc. Natl Acad. Sci. USA 89(7): 2624-2628). Seeds from both B. campestris and B. napus plants were produced by antisense expression of a cDNA encoding the B. campestris stearoyl-ACP desaturase, the enzyme responsible for introducing the first double bond into 18 carbon fatty acids in plants, using a seed specific promoter region. These seeds produced oils high in stearic acid, that also contained elevated levels of linolenic acid (18:3), when compared to seeds from unmodified plants from the same species. Elevated levels of stearic acid have been obtained in soybean by a similar underexpression of stearoyl-ACP desaturase (U.S. Pat. No. 5,443,974) and in canola by over expression of an acyl-ACP thioesterase (U.S. Pat. No. 5,530,186). Mutation breeding has also produced soybean lines with elevated levels of stearic acid in their seed oils (Graef, G. L. et al., (1985) JAOCS 62:773-775; Hammond, E. G. and W. R. Fehr, (1983) Crop Sci. 23:192-193).
Poly-unsaturated fatty acids contribute to the low melting point of liquid vegetable oils. In high saturate oils their presence is a detriment in that they decrease the melting point, and therefore even higher levels of undesirable saturated fatty acid are required to achieve a plastic fat at room temperature. Additionally, when used in baking and confectionery applications high levels of poly-unsaturates leads to oxidative instability as described above for liquid oils. Thus for maximum utility a high saturate fat produced in soybean should contain saturated fatty acids, mono-unsaturated fatty acid and as little poly-unsaturated fatty acid as possible. Gene combinations discovered in this invention provide novel fatty acid profiles in soybean which meet these criteria.